The central role of the macrophage (Mx) in Fe homeostasis and in the sideropenic anemias associated with inflammation is recognized but the process is poorly understood. We have, therefore, characterized human Mx hemoglobin (Hgb)-Fe metabolism in vitro. Human monocyte (mo) monolayers were used either within 24 hr or matured into Mx by culture for 14 days. Mo or Mx phagocytized 59Fe-Hgb labelled murine erythrocytes (rbc) for 1 hr. to assess 59Fe release, extracellular rbc were then lysed and media with 40% autologous serum was added. Culture media and Mx lysates were separated by acid precipitation or Sephadex-DEAE salt gradients. Mx phagocytized more rbc than Mo: 18.3 plus or minus 3.0 x 10 th the 5th power (mean plus or minus SE) rbc/10 to the 5th power Mx vs. 5.2 plus or minus 1.9 x 10 to the 5th power rbc 10 to the 5th power Mo, n equals 13, P is less than .01. After 72 hr (37 degrees Centrigrade, 4 % COx) Mx released 4.0-32.2% (13.4 plus or minus 3.1) of the initially phagocytized rbc-Hgb-59Fe into the supernatant media transferrin pool; this release was inversely proportional to the number of rbc initially phagocytosed (r equals 0.72, n equals 24, P is less than .01). Mo released a small fraction of phagocytized 59Fe (3.7 plus or minus 1%). The rate of Mx 59Fe released to the supernatant transferrin pool decreased with time: the Fe from 0.46 plus or minus .13 micrograms Hgb/10 to the 5th power Mx in the first hr, 0.22 plus or minus .04 micrograms hgb/hr/ 10 to the 5th power Mx from 2024 hr, and .02 plus or minus .01 micrograms Hgb/hr/10 to the 5th power Mx from 24-72 hr. Release was abrogated at 4 degrees Centigrade. Supernatants from phagocytizing granulocytes inhibited early phase release (33% P is less than .01). Intracellular Mx Hgb-59Fe metabolism was examined by fractionating Mx monolayer lysates 1-72 hours after rbc phagocytosis. With increasing time less 59Fe was associated with Hgb, and more with ferritin: Hgb%/ferritin % equals 52/32, 3/78, 0.4/93, 0/95 at 1,3,24,72 hr respectively. Other 59Fe eluted with transferrin. This in vitro characterization of human Mx Hgb-Fe metabolism suggests that this system will be useful in elucidating Mx dysfunction in certain disorders of Fe homeostasis.